The present invention relates to methods for modulating immune coagulation using novel antibodies and compounds that modulate immune coagulation.
Activation of the coagulation pathways is an important part of immune and inflammatory reactions and is associated with bacterial and viral infections (e.g. endotoxin shock, viral hepatitis), glomerulonephritis (GN), cancer, a number of gastrointestinal diseases, allograft and xeno graft rejection and spontaneous or stress-triggered fetal loss. Immune coagulation is mediated by a number of coagulants that, when triggered, activate specific ligands resulting in cleavage and activation of coagulation pathways that lead to fibrin deposition. The molecular events leading to expression of immune coagulants involve natural antibodies binding both to antigens on endothelial cells and Fc receptors on macrophages and endothelial cells. An additional mechanism is immune complex-mediated induction of macrophage procoagulants. These events lead to thrombin production which initiates platelet activation and ultimately fibrin deposition.
In 50% of hepatitis patients moderate to severe consumptive coagulopathy or disseminated intravascular coagulopathy is found associated with fulminant hepatitis. Thrombi formation is observed around necrotic areas (Sinclair et al., 1990 and Lee, W. M., 1993). As a consequence of hepatitis, levels of factors II, V, VII, and X are decreased in the liver, reflecting both consumptive coagulopathy and a decrease in hepatic synthetic function. Also, the levels of thrombin-antithrombin complexes are high and platelet counts are low (Lee, W. M., 1993). These results indicate that the host immune system, including the coagulation pathway, is disrupted as a result of HBV infection. The limited host range of HBV and the difficulty to propagate the virus in tissue culture have hampered the understanding of HBV and hepatitis B.
Mononuclear phagocytes and macrophages are implicated in the pathogenesis of hepatitis specific induction of procoagulant activity because of their role in coagulation; they synthesize some of the essential coagulation factors such as tissue factor and their surfaces serve as sites of fibrin deposition. Factors participating in the coagulation cascade are released as inactive zymogens and upon activation, by preceding activated factors, they are converted to their active form. The factors are predominantly serine proteases (Davie et al., 1991). Factors VIIa, XIIa, XIa, Xa, IXa, thrombin, kallikrein, and plasminogen are categorized under family 1 serine proteases (Davie et al., 1991; Barrett and Rawlings, 1995; Rawlings and Barrett, 1994; Nduwimana et al., 1995). In order to initiate the coagulation cascade the procoagulants need to be expressed. Ruegg and Pytela, 1995 isolated a cDNA encoding a protein that is homologous to a murine fibrinogen-like protein (Koyama et al. 1987). However, they did not determine the function of the protein or realize its use in modulating immune coagulation.
In view of the many diseases associated with the activation of the coagulation pathways, there is a need to identify and characterize procoagulants and to develop methods for modulating immune coagulation that are useful in the prevention, treatment and diagnosis of diseases associated therewith including bacterial and viral infections, glomerulonephritis (GN), cancer, a number of gastrointestinal diseases, allograft and xenograft rejection and spontaneous or stress-triggered fetal loss.
The present inventor has identified and characterised an immune procoagulant, and the molecular and cellular events leading to its production. Specifically, the mouse and human direct prothrombinase genes (referred to herein as xe2x80x9cmFgl2xe2x80x9d and xe2x80x9chFgl2xe2x80x9d respectively) have been cloned and sequenced. The nucleic acid sequence of the human and mouse Fgl2 is shown in SEQ.ID.NOS.:1 and 3, respectively. The genes encode a transmembrane serine protease which has functional prothrombinase activity. The proteins encoded by the genes have been sequenced in both humans and mice. The protein has a molecular weight of approximately 70 kd. The hfg12 gene has been mapped to chromosome 7 and the mFgl2 gene to chromosome 5. The inventor has cloned and sequenced the genomic DNA encoding the human prothrombinase. The organization of the genomic DNA encoding hFgl2 is shown schematically in FIG. 1. The nucleic acid sequence of the promoter region, exon 1, exon 2 and the 3xe2x80x2 UTR are shown in FIGS. 8, 2, 3 and 4, respectively. The amino acid sequence of the human and mouse Fgl2 protein is shown in FIG. 5 and in SEQ.ID.NOS.:2 and 4, respectively.
The determination by the inventor that Fgl2 is a direct prothrombinase allows the development of diagnostic methods and therapies for conditions involving immune coagulation.
Accordingly, the present invention provides a method of inhibiting immune coagulation comprising inhibiting the activity or expression of Fgl2. The method can be used in viva to treat a condition which requires a reduction in immune coagulation such as bacterial and viral infections, glomerulonephritis (GN), cancer, a number of gastrointestinal diseases, allograft and xenograft rejection and fetal loss.
In one aspect, the activity of Fgl2 may be inhibited using an antibody that binds to Fgl2. The present inventor has developed a pariel of monoclonal and polyclonal antibodies which neutralize Fgl2 and prevent the fibrin deposition associated with endotoxin shock, viral hepatitis, allograft and xenograft rejection. The antibodies were shown to prevent cellular infiltration, fibrin deposition and tissue damage, and lead to enhanced survival. In particular, antibodies against the direct prothrombinase (Fgl2) were found to be extremely useful in preventing diseases known to have associated massive fibrin deposition and coagulative necrosis, including allograft and xenograft rejection as well as fetal loss induced by stress or cytokines.
In one embodiment, the present invention provides a method of preventing or reducing graft rejection comprising administering an effective amount of an antibody to Fgl2 to an animal in need thereof.
In another embodiment, the present invention provides a method of preventing or reducing fetal loss comprising administering an effective amount of antibody to Fgl2 to an animal in need thereof.
Antibodies can be prepared using entire Fgl2 proteins or immunogenic portions thereof. Preferably, such portions bind with an affinity of at least about 106 L/mole to an antibody raised against Fgl2. In particular, the present inventor has shown that a peptide comprising amino acid residues 300 to 400 is useful in raising antibodies. Accordingly, the present invention contemplates antibodies which (a) immunoreact with peptides comprising the amino acids at approximately positions 300 to 400 in FIG. 5; and (b) neutralize the prothrombinase activity of hFgl2. The invention also relates to hybridoma cell lines that produce the monoclonal antibodies, and inhibitors and activators thereof.
In another aspect, the expression of Fgl2 may be inhibited using antisense molecules that are complimentary to a nucleic acid sequence from the Fgl2 gene. In particular, the nucleic acid sequences for Fgl2 as shown in FIGS. 2 or 3 may be inverted relative to their normal presentation for transcription to produce antisense nucleic acid molecules.
Additional inhibitors of Fgl2 may be identified by testing substances that inhibit the prothrombinase activity of Fgl2. In particular, the invention contemplates a method for assaying for a substance that affects the prothrombinase activity of Fgl2 comprising (a) reacting Fgl2, a substrate which is capable of being cleaved by Fgl2 to produce a product, and a test substance, under conditions which permit cleavage of the substrate to produce the product; (b) assaying for product; and (c) comparing to the product obtained in the absence of the substance to determine the affect of the substance on the prothrombinase activity of the Fgl2 protein.
The nucleic acid molecules encoding Fgl2, Fgl2 proteins, and monoclonal antibodies of the present invention have diagnostic and monitoring applications. In particular they may be used in conventional assays to monitor or diagnose conditions such as bacterial and viral infections (e.g. endotoxin shock, viral hepatitis), allograft rejection, glomerulonephritis, cancer, a number of gastrointestinal diseases and fetal loss.
In one embodiment, the present invention provides a method for diagnosing or monitoring a condition involving increased immune coagulation in an animal comprising detecting a Fgl2 protein or a Fgl2 nucleic acid in a biological sample from the animal.
The invention also contemplates compositions comprising, and methods of using (a) the monoclonal antibodies produced by the hybridoma cell lines of the invention; (b) inhibitors and activators of the monoclonal antibodies; (c) antibodies to a Fgl2; (d) antisense nucleic acid molecules to fg12; and (e) substances identified using the methods of the invention (e.g. inhibitors and activators of the expression of a nucleic acid molecule of the invention; and, inhibitors and activators of the activity of a Fgl2 protein of the invention).
The compositions of the invention may be used in the prevention or treatment of conditions requiring a reduction in procoagulant activity. Therefore, the invention contemplates a composition for treating a condition requiring a reduction m procoagulant activity comprising administering a therapeutically effective amount of one or more inhibitors of Fgl2. The inhibitor may be an antibody specific for a Fgl2; an antisense nucleic acid molecule of the invention; substances identified in accordance with the methods of the invention or a monoclonal antibody produced by a hybridoma cell line of the present invention. Conditions which require reduction in procoagulant activity include bacterial and viral infections (e.g. endotoxin shock, viral hepatitis), allograft and xenograft rejection, glomerulonephritis, cancer, a number of gastrointestinal diseases and fetal loss.
The present invention also contemplates a vaccine for preventing graft rejection comprising an amount of a Fgl2 protein which is effective to provide protection against graft rejection.
The present invention also contemplates a vaccine for preventing fetal loss comprising an amount of a Fgl2 protein which is effective to provide protection against fetal loss.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.